PROJECT 2 SUMMARY/ABSTRACT Checkpoint blockade has thus far demonstrated clinical benefit in only a small fraction of pediatric cancers and emerging paradigms hold that this is largely due to their low mutational burdens. In contrast, CAR T cells directed toward B cell restricted antigens have demonstrated astonishing potency against B-ALL, despite low mutational burden. The recent FDA approval of tisagenlecleucel (Kymriah) for pediatric and young adult B-ALL is a watershed moment, representing the first cell based therapy approved for the treatment of cancer and the first gene therapy approved in the United States. Clinical and preclinical data with non-CD19 CAR T cells is providing increasing evidence that CAR therapeutics will demonstrate activity in AML, solid tumors and brain tumors in the coming years, driven largely by technological innovations in CAR engineering that will create safer and more potent therapeutics. This Project leverages advances in technology to improve fundamental understanding of the major factors currently limiting CAR efficacy and to develop next generation CAR T cells, designed specifically to overcome these challenges. Work on each aim will launch simultaneously and be conducted in parallel, however information gleaned in separate aims will directly inform the others during the course of the Project and enhance Project deliverables. Crosstalk with other Projects in the Center will be continual and extensive as described below. A major goal of our Center is provision of a comprehensive catalog of cell surface molecules expressed on high risk pediatric tumors in Project 1, and identification of ?antigen groups? that traffic together, driven by commonalities in genotype, microenvironment and/or cell lineage. In Aim 1, we will utilize MIBI and CyTOF to undertake ultra-high dimensional analysis of high risk pediatric cancers, and datasets generated will be interrogated by experts in computational science with Project 1 using algorithms equipped to identify patterns of antigen expression within histologically or genotypically associated groups, and inform approaches to develop multi-specific CAR T cells in Aim 2. Here, we leverage advances in in silico modeling and super resolution subcellular imaging to optimize engineering of these complex receptors, and we also test whether targeted gene integration, using CRISPR-Cas9 will render more potent multi-specific T cells than the traditional approach using retroviral/lentiviral vectors. Aim 3 takes a deep dive into the pathobiology of T cell exhaustion in CAR expressing T cells, and leverages innovative technologies for discovery (ATAC-Seq) and engineering (drug regulatable proteins) to create the first clinically applicable class of regulatable (e.g. remote controlled) CAR T cells. There will be substantial crosstalk between Aim 2 and Aim 3, as engineering advances used to overcome exhaustion can be readily applied to multi-specific CARs to further enhance potency. Further, this Project will interface substantially with Project 3, since delineation of major elements comprising the TME delivered by MIBI analysis of pediatric tumors, will inform selection of immunomodulators for study in the Project 3?s fully murine tumor models.